Changes in cell monolayer barrier integrity and compromised barrier function are key features of many pathologic conditions including atherosclerosis (Hirase and Node, 2012), cancer (Le Guelte et al., 2011), stroke (Fraser, 2011), inflammation, pulmonary edema (Fishel et al., 2003; Ware and Matthay, 2005) and others. The methods for in vitro evaluation of mass transport across the cell monolayer have wide applications in studies addressing mechanisms of cell monolayer integrity, regulation of blood-gas, blood-brain, and other barriers, as well as in drug discovery research.
Existing approaches evaluate monolayer permeability directly by measuring the amount of a labeled macromolecular tracer traveling through a cell monolayer grown on a porous filter (transwell permeability assays), or indirectly by measurements of electrical resistance across the cell monolayer under different stimulation conditions (Balda et al., 1996; Giaever and Keese, 1984). The advantage of the transwell permeability assay is its ability to test the size selectivity of intercellular barriers. Limitations include relatively low sensitivity, considerable time between measurements, low throughput format of assay, absolute requirement of complete coverage of transwell membrane by cell monolayer, fluid convection factor and diffusion characteristics of transwell membrane materials which can mask minor changes in monolayer barrier function of endothelium (Lo et al., 1999).
The advantage of electrical resistance measurements across a cell monolayer is the ability of data acquisition in real time and high sensitivity of the method. This technique however requires high cost equipment, provides indirect means for permeability evaluation, and usually uses a limited area of cell monolayer for permeability analysis.
While each existing method offers particular advantages, none allow for spatial resolution of local changes in permeability representing regional variations of endothelial and epithelial barriers in vivo, as reflected by studies of regional heterogeneity of lung injury in vivo (McVerry et al., 2004). Existing assays are also unable to evaluate permeability in endothelial or epithelial monolayers grown on elastic substrates and exposed to cyclic mechanical stretch, a key feature of ventilator induced lung injury and pathologies associated with over-distension of other organs. Embodiments described below provide for a new measurement and visualization of local permeability in cell monolayers exposed to variety of a chemical and/or mechanical stimuli.